And NADP-Dependent Alcohol Dehydrogenase Enzymes of Strawberries
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J. AMER. SOC. HORT. SCI. 120(5):798-801. 1995. Characterizing NAD- and NADP-dependent Alcohol Dehydrogenase Enzymes of Strawberries William C. Mitchell1 and Gojko Jelenkovic Plant Science Department, Rutgers University, New Brunswick, NJ 08903 Additional index words. Fragaria ×ananassa, fruit ripening, isozymes, organoleptic quality Abstract. The NAD-dependent and NADP-dependent alcohol dehydrogenase activities of strawberries (Fragaria xanan- assa Duch.) were found to have broad substrate specificities including those alcohols and aldehydes responsible for strawberry aroma and flavor either directly or through their ester products. NAD-dependent activities were greatest against short-chained alcohols, whereas the NADP-dependent activities were most active against aromatic and terpene alcohols. Differences were seen in substrate specificity between receptacle and achene alcohol dehydrogenase activities. Alcohol dehydrogenase activities were found to be developmentally regulated in receptacle tissue and increased during the period of fruit maturation and ripening. Isoelectric focusing of NAD-dependent ADH activities showed that several isozymes of this enzyme exist, that they differ between receptacle and achene tissues, and that they vary among specific genotypes. Our results suggest that NAD- and NADP-dependent ADH activities are integral components of flavor and fragrance volatile production in ripening strawberries. NAD-dependent (NAD-ADH; E.C. 1.1.1.1) and NADP-depen- Materials and Methods dent alcohol dehydrogenases [NADP-ADH (cinnamyl alcohol dehydrogenase); E.C. 1.1.1.2] exist in a wide variety of higher Plant material. Primary fruit were freshly harvested from straw- plant tissues (Davies et al., 1973; Freeling and Bennett, 1985; berry plants that were either grown in a greenhouse (genotype NJ Hatanaka et al., 1973; Kanellis et al., 1991; Noueiry, 1989; Roe et 8343-6) or field (NJ 8336-1, NJ 8611-1, ‘Earliglow’, ‘Raritan’). al., 1984; Tress1 and Albrecht, 1986; Yamashita et al., 1978). Four developmental stages of strawberries were used in this Putative roles attributed to plant alcohol dehydrogenases include study. Stage 1 and stage 2 fruit had fully white receptacles and surviving periods of hypoxia (Freeling and Bennett, 1985; Kanellis green achenes. Stage 3 fruit had receptacles with pink and white et al., 1991; Roe et al., 1984), regulation of development (Mitchell sections and green achenes. Stage 4 fruit were fully red, fragrant, and Jelenkovic, 1992; Roe et al., 1984), production of cinnamyl and had brown achenes. Average fresh weights of stage 1 through alcohols for the lignification of cell walls (Davies et al., 1973), stage 4 fruit were 0.315 ± 0.056 g, 1.204 ± 0.106 g, 6.492 ± 1.236 protection from chilling (C. Frenkel, personal communication), g, and 8.302 ± 2.015 g, respectively. and biogenesis of flavor and fragrance volatiles (Hatanaka et al., Protein extraction from achenes. Protein extractions from 1973; Noueiry, 1989; Tress1 and Albrecht, 1986; Yamashita et al., achenes and receptacles were performed at 4C. Achenes were 1978). removed from ripe strawberries, washed to remove debris, and air Biosynthetic pathways of alcohols, aldehydes, and esters impli- dried. Achene tissue was ground in 1.5-ml microfuge tubes with cated in flavor and fragrance production have been tentatively tapered glass rods in an extraction buffer composed of 100 mM determined for many fruit (Tress1 and Albrecht, 1986). In straw- Tris-HCl, pH 7.5, 2% w/v soluble polyvinylpyrrolidone (PVP) as berries, exogenously applied fatty acids, aldehydes and alcohols a phenolic scavenger, 1 mM ZnSO4, 10 mM b- mercaptoethanol (Yamashita et al., 1977; Yamashita et al., 1979) were metabolized (BME) as a reducing agent, and the protease inhibitors 0.8 mM to their corresponding alcohols and esters. These results suggested phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine hy- that endogenous alcohols and aldehydes are interconverted by drochloride (BHC), and 5 mM e- amino-n-caproic acid (ACA). 100 ADH in the pathway for producing the complex mix of volatile µl of buffer was used per 10 mg of fresh weight. The extract was compounds (Honkanen and Hirvi, 1990) responsible for the char- centrifuged for 15 min at 13,600× g and the supernatant was acteristic flavor and fragrance of strawberries. collected as the enzyme source. The present work examines the substrate specificity of straw- Protein extraction from receptacles. Strawberries were har- berry ADH activities, the isozyme pattern of the NAD-dependent vested and achenes were removed from the receptacles by peeling ADH, and changes in ADH activities that occur during fruit the epidermis away with razor blades. Achene-free receptacles ripening and development. Our results support the hypothesis that were then homogenized in an extraction buffer composed of 100 NAD-dependent and NADP-dependent ADH enzymes are impor- mM Tris-HCl (pH 8.5), 2% w/v PVP, 20 mM CaCl2, 0.1 mM ZnSO4, tant components of the in vivo biosynthetic pathway of strawberry 0.8 mM PMSF, 1 mM BHC, 5 mM ACA, and 10 mM BME in a food flavor and fragrance volatiles. blender. The extract was filtered through one layer of Miracloth (Calbiochem) and the pH of the supernatant was adjusted to 7.5. The extract was then stirred for 15 min and centrifuged at 30,000× Received for publication 18 July 1994. Accepted for publication 10 Apr. 1995. This g to remove insoluble residues. The supernatant was made to 80% research was supported by grant no. 12-113 from the New Jersey Agricultural saturation in ammonium sulfate, stirred for 30 min, and centri- Experiment Station. We thank Chaim Frenkel and Joseph Goffreda for their critical fuged 20,000× g for 15 min to precipitate protein. The pellet was review of the manuscript. Use of trade names does not imply endorsement of the resuspended in a minimal volume of a buffer composed of 100 mM products named nor criticism of similar ones not named. The cost of publishing this Tris-HCl (pH 7.5), 0.1 mM ZnSO , 0.8 mM PMSF, 1 mM BHC, 5 mM paper was defrayed in part by the payment of page charges. Under postal regula- 4 tions, this paper therefore must be hereby marked advertisement solely to indicate ACA, and 10 mM BME and dialyzed against 50 mM Tris-Cl (pH this fact. 7.5), 0.1 mM ZnSO4, 0.8 mM PMSF, 1 mM BHC, 5 mM ACA, and 1 Present address: Biology Dept., Jersey City State College, Jersey City, NJ 07305. 10 mM BME. 798 J. AMER. SOC. HORT. SCI. 120(5):798-801. 1995. The protein extraction method for developmental studies was used when comparing relative activities for different substrates simpler. Achenes were removed from receptacles as above and the (Tables 2 and 3), except for the substrates cinnamyl alcohol (20 achene-free receptacles were homogenized in a Potter-Elvehjem mM) and geraniol (5 mM), which have relatively poor solubility in tissue grinder, in an extraction buffer composed of 100 mM Tris- aqueous solutions. Calculation of NAD(P) formed was assessed by HCl (pH 8.5), 2% w/v PVP, 0.1 mM ZnSO4, 0.8 mM PMSF, 1 mM the change in A340 (Worthington, 1988). Preliminary results with BHC, 5 mM ACA, and 10 mM BME. The extract was adjusted to pH strawberry NAD-dependent ADH gave a KM with ethanol as 7.5 and centrifuged at 13,600× g for 15 min to pellet insoluble substrate of 6.96 ± 0.32 mM, and strawberry NADP-dependent debris. The supernatant was used as the enzyme source. ADH gave a KM with benzyl alcohol of 24.82 ± 1.28 mM. Our KM ADH assays. All ADH assays were carried out at 24C. Oxida- values were similar to those of other plant NAD- and NADP- tions of alcohols by ADH were assayed in reaction mixes contain- dependent ADH enzymes (Bicsak et al., 1982; Davies et al., 1973; ing 100 mM glycine-KOH (pH 9.5) and 1.3 mM NAD or NADP, Roe et al, 1984; Yamashita et al., 1978). while reductions of aldehydes were assayed in reaction mixes Native isoelectric focusing. Native isoelectric focusing (IEF) containing 100 mM Tris-HCl (pH 7.5) with 1.3 mM NADH or was performed on a horizontal flat bed Fisher Biotech FN 1001 IEF NADPH. Substrate concentrations of 100 mM were used to devel- system according to manufacturers instructions. Gels 1 mm thick opmental course assays. Substrate concentrations of 40 mM were were composed of 1% (w/v) agarose, 13% (w/v) sorbitol, 6.7% (v/ Table 1. ADH activities of stage four strawberries.Z “Activities were determined for primary fruit of genotype NJ 8343-6. NAD-ADH activity was measured using 100 mM ethanol as substrate, and NADP-ADH activity was measured using 100 mM benzyl alcohol as substrate. Table 2. Substrate specificity of strawberry NAD-ADH.z “Specific activity for NAD-ADH (genotype NJ 8336-l) was measured using 40 mM substrate except in the cases of cinnamyl alcohol and geraniol, where 20 mM and 5 mM were used, respectively. Values are ± SE. Table 3. Substrate specificity of strawberry NADP-ADH.z zSpecific activity for NADP-ADH (genotype NJ 8336-l) was measured using 40 mM substrate except in the cases of cinnamyl alcohol and geraniol, where 20 mM and 5 mM were used, respectively. Values are ± SE. J. AMER. SOC. HORT. SCI. 120(5):798-801. 1995. 799 v) ampholines (pH 4 to 6.5), and 1% (v/v) Triton X-100 (Fisher). Isozyme differences may account for the differences in substrate The electrode buffers were 0.05 N H2SO4 and 1 N NaOH. Electro- specificity we have seen between our achene NAD-dependent phoresis was carried out with 15 W constant power for 2 h at 9C. ADH and that of Yamashita et al. (1978) since the plants were After electrophoresis, gels were stained for NAD-dependent ADH genetically different.